Frequency and clinical impact of viraemia in paediatric patients undergoing therapy for cancer

In contrast to transplant recipients, there is a paucity of data regarding frequency and clinical significance of viraemia in children receiving conventional chemotherapy. In a prospective observational study, we assessed the frequency of and clinical impact of viraemia with cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, human herpesvirus-6 (HHV6) and herpes-simplex virus 1/2 (HSV1/2) in paediatric cancer patients at diagnosis, at a routine examination during intensive chemotherapy, and during febrile neutropenia (FN). Seventy-nine patients (median age 6 years; 66 children with haematological malignancies) were included in the study. Overall, 362 blood samples were analysed, 72 from the time at diagnosis (11.1% with positive PCR result), 118 during a regular control after chemotherapy (11.0% positive), and 159 during FN (8.8% positive). The overall positivity rate was 9.6% (CMV 3.3%, HHV6 2.7%, HSV 2.2%, EBV 0.8% and adenovirus 0.3%). There were no significant differences between FN episodes with and without viraemia in terms of duration of fever or neutropenia/lymphopenia, severity of mucositis (> II0), incidence of diarrhea and ICU admission. Our results indicate that viraemia in paediatric cancer patients generally does not have a major clinical impact, and may help in the decision regarding the indication of routine evaluation for viraemia in febrile neutropenic, but otherwise asymptomatic children.


Definitions
Fever was considered as temperature > 38.5 °C once or 38.0-38.5 °C twice within a 1-h interval, and neutropenia as an absolute neutrophil count < 500/μl.Lymphopenia was defined according to age-dependent reference values given by Tosato et al. 8 .Mucositis was measured using the Oral Assessment Guide (OAG) 9 .One cycle of chemotherapy was defined as the time from the start of chemotherapy until the day before the start of the next cycle of chemotherapy.

Study design
Clinical and laboratory data were collected throughout intensive chemotherapy, and the viral load for CMV, HSV1/2, HHV6, EBV, and adenovirus was assessed in the plasma at several time points.First, the viral load was evaluated at the time of diagnosis of the malignancy.At that time, also specific antibody titres against CMV, HSV1/2, and EBV were determined, whereas antibody titres for HHV6 and adenovirus were not regularly assessed.Second, blood was evaluated for viraemia at a routine control after both the second and fourth cycle of chemotherapy, when the child presented without infectious complication ("regular control during chemotherapy").Last, blood was assessed when the patient presented with fever during neutropenia, irrespective whether a bacterial pathogen or another source of infection had been identified ("febrile neutropenia").

Virological tests
DNA was extracted from 500 µl plasma sample using Qiasymphony DSP virus/pathogen midi kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer´s instructions.CMV viral load was estimated as previously described 10 .The lower limit of CMV quantification was 200 IU/ml, and positive qualitative results of tests below this limit were indicated as < 200 IU/ml.Extracted DNA was also used for semi-quantitative determination of adenovirus, HSV1/2, HHV6 and EBV-specific DNA 11,12 .According to the results of ring-tests (inter-laboratory tests) to evaluate the reproducibility of virus detection between different laboratories, the limit of quantification was 500 copies/ml for all assays.Copy numbers of the different viruses were estimated using these tests results, whereas positive qualitative results of tests below the limit of quantification were indicated as < 500 copies/ml.Specific primer und probes used are listed in Supplement Table 1.
As this was an exploratory study and in order to avoid that viral test results will influence a clinical decision regarding antiviral treatment, all serum samples for PCR testing were frozen at − 20 °C and kept until the patient had completed intensive chemotherapy.

Statistical analysis
Data were analysed using GraphPad Prism (version 5.04; Graph-Pad Software; https:// www.graph pad.com/).Data on PCR results in the different populations and time points were reported in a descriptive way.For comparing febrile neutropenic episodes in patients with positive viral PCR to those with negative results of viral PCR, the Mann-Whitney U test (for comparison of the median) or Chi-square-test or Fisher's exact test was used.Only the first episode of reactivation with a specific virus during febrile neutropenia was included in the analysis, as repeated episodes of reactivation with the same virus were not considered to be independent events.All statistical tests were two-sided, and a P value of < 0.05 was considered as significant.
Fifty-seven patients (72.2%) did not have any positive PCR result during the study period.Out of these, 17 (29.8%),21 (36.8%) and 22 (38.5%)patients had a positive test result for specific IgG against CMV, HSV1/2 and EBV, respectively, at the time of the diagnosis of the malignancy.Twenty-two patients (27.8%) had at least one positive PCR result during the study period, with positive results for specific IgG against CMV, HSV1/2 and EBV in 9 (40.9%),7 (31.8%)and 9 (40.9%) of the patients, respectively (no significant difference between patients with specific IgGs who had or did not have a positive PCR during the study period).In 9 patients with ALL (25% of patients with ALL), 3 with AML (18.8%), 5 with NHL (35.7%), and 5 with neuroblastoma (50%) a positive sample for viraemia was found during the study period, whereas none of the patients with Ewing sarcoma had a positive PCR result.Thirteen of the patients had one single positive PCR result during the study period (4 patients prior to therapy (CMV n = 2, HSV1/2 1, EBV 1), 6 patients at a regular control during chemotherapy (HSV1/2 4, CMV 1 und HHV6 1), and 3 patients during febrile neutropenia (CMV 2, HSV1/2 1)].In contrast, PCR testing revealed more than one positive result during the study period in 9 patients: a positive PCR result with the same virus was seen in 5 patients (three patients with HHV6, two with CMV), and different viruses were detected in 4 patients.

Discussion
While infections with viruses such as CMV, HSV or adenovirus are a common and often severe problem in children after HCT and mandate the institution of antiviral therapy in many patients, the frequency and clinical significance of a positive viral PCR in the blood of children receiving conventional chemotherapy is relatively unclear.In our prospective monocentre study, a total of 9.3% of the 362 blood samples were positive for one of the viruses tested, namely in 3.3% for CMV, 2.7% for HHV6, 2.2% for HSV1/2, 0.8% for EBV and 0.3% for adenovirus, respectively.Overall, 27.8% of the patients had at least one positive PCR finding during intensive chemotherapy.A study in 77 children receiving chemotherapy for a variety of underlying malignancies reported similar findings for CMV and EBV with 3.4% and 0.5% of positive samples, respectively, but also observed that 9.3% of samples were positive for HHV6, which is considerably higher compared to our results 13 .Published data demonstrate a wide variation in the prevalence of viraemia in patients treated for cancer, which, for example, ranges from 2.5% to 52% for CMV or from 5.9% to 26.3% for HHV6 [14][15][16] .These differences might be explained by several factors, such as by differences in the age range of tested patients, different methods of virus detection and different thresholds of positivity 14,[16][17][18] .Due to different sampling strategies (e.g., screening every two weeks throughout intensive chemotherapy versus sample collection only during prolonged febrile neutropenia) resulting in a wide variation of the number of samples (between 141 and 1,223), a meaningful comparison of the studies regarding the percentage of patients with viraemia cannot be made 13,15 .Corroborating our data, no major impact of the underlying malignancy on the incidence of viraemia had been observed in other studies 13,16 .Our observation that there were no differences in overall frequencies of viral detection between patients who were seropositive or seronegative at diagnosis is different from previous studies and might be explained by differences in the prevalence of seropositivity and by the small sample size 15,17 .We did not find a significant difference of the frequency of viraemia between test results prior to chemotherapy, test results during a routine control and those during an episode with febrile neutropenia, which were 11.1%, 11.0% and 8.8%, respectively.Similar results were observed by Goldfarb et al. with 11.5% and 9% of positive results in acute and routine visits, respectively 13 , whereas others reported that the detection of HHV6 in blood doubled from initial assessment to testing during a febrile episode (17.4% to 37.1%) 18 .Notably, one study reported that the majority of CMV reactivation in ALL www.nature.com/scientificreports/patients occurred during maintenance therapy, a time period which was not included in our analysis 19 .This observation can be explained by the fact that maintenance therapy in ALL consists of daily 6-mercaptopurine and weekly methotrexate, which leads to prolonged lymphopenia, and low lymphocyte counts have been described as a risk factor for CMV reactivation 16 .Severe HHV6 infections have been observed in up to 6% of cancer patients during treatment with cytotoxic chemotherapy, and HHV6, in particular during a coinfection with CMV, significantly increases the risk for serious clinical complications, such as bacterial and fungal infections 16,18 .Importantly, also fatal events due to virus infection have been described in children receiving therapy for cancer 20 .On the other hand, it is currently unclear whether viraemia in febrile neutropenic children who are otherwise asymptomatic has clinical consequences and patients benefit from antiviral therapy, even in case of high copy numbers.For example, in one study, 7 asymptomatic children suffering from ALL revealed high level of CMV viraemia 15 .According to the physician´s discretion, four of them were treated with intravenous ganciclovir with or without subsequent oral valganciclovir, whereas three patients did not receive antiviral treatment.Whether treated or not, all patients remained well during the entire study and no end-organ diseases were detected.In our analysis, we did not observe any severe clinical complications due to virus infection, and in none of the patients, organ involvement (e.g., retinitis, pneumonia, hepatopathy) occurred.In addition, none of the patients received preemptive antiviral therapy.However, we observed only low levels of viraemia in the patients, which is similar to the results of other studies 14 .In addition, in comparison with case-matched controls without viraemia we found that children receiving chemotherapy and having viraemia do not experience more days of neutropenia or of lymphopenia, and do not suffer from longer duration of fever, higher grade of mucositis, higher incidence of diarrhea or ICU admission than children without positive PCR results.Corroborating our data, others did not find an association between HHV6 viraemia and individual acute clinical complications, and therefore, clinical events may be misattributed to the virus 13 .Based on the current data and the fact that many antiviral drugs are associated with severe potential adverse events, we do not preemptively treat even high level viraemia in asymptomatic paediatric patients receiving chemotherapy for an underlying malignancy, which is in contrast to the setting of HCT 21 .In addition, we do not screen febrile neutropenic, but otherwise asymptomatic patients for viraemia, which has been suggested by some experts 14,19 .
We recognise that our study has some important limitations.Although this is one of the largest prospective studies in children, it is a monocentre approach which does not allow to generalise the findings.In addition, due to the irregularity of sampling intervals, we might have missed positive findings, which could be associated with clinical complications.Last, we did not analyse the time of maintenance therapy, which might have higher incidence rates of viraemia 15,19 .On the other hand, our study is unique as all virological testing of blood samples was performed after the end of intensive chemotherapy of a specific patient, and therefore, data and in particular treatment decisions are unbiased.
In conclusion, our data need to be validated in other centres and with other therapy protocols, but our results indicate that viraemia in paediatric cancer patients receiving intensive chemotherapy generally does not have a major clinical impact, and may help in the decision regarding the indication of routine evaluation for viraemia in febrile neutropenic, but otherwise asymptomatic children.

Table 1 .
Patients´ characteristics and number of positive PCR results at the time of diagnosis, during a regular control after chemotherapy and during an episode of febrile neutropenia.ALL acute lymphoblastic leukemia, AML acute myeloid leukemia, NHL non-Hodgkin lymphoma, EBV Epstein-Barr virus, HHV6 human herpesvirus-6; CMV cytomegalovirus, HSV1/2 herpes simplex virus ½. *viral load for CMV expressed in IU/ ml, for all other viruses in copies/ml.

Table 2 .
Patients with viraemia during an episode of neutropenia.NB neuroblastoma, AML acute myeloid leukemia, ALL acute lymphoblastic leukemia, MR intermediate risk, HR high risk, f female, m male, neg negative, pos positive, n.d.not done, EBV Epstein-Barr virus, HHV6 human herpesvirus-6, CMV cytomegalovirus, HSV herpes simplex virus, IgG immunoglobulin G. *prior to diagnosis of malignancy.*viral load for CMV expressed in IU/ml, for all other viruses in copies/ml.